The CFTR protein forms a chloride ion channel in the plasma membranes of many epithelial cells, including cells of the kidney and gut. Mutation of the gene encoding CFTR is the primary defect in Cystic Fibrosis (CF), the most common lethal, autosomal recessive disease among Caucasians, affecting approximately 30,000 Americans. Alteration in CFTR function also plays an important role in the pathophysiology of secretory diarrhea and polycystic kidney disease (PKD). The basic mechanisms of permeation in this channel are not clear. It is not known which portions of the protein contribute to forming the pore, and which amino acids in those domains are involved in the biophysical processes of ion permeation. The long-term objective of this laboratory is to determine the mechanisms of permeation in CFTR. For this proposal, Specific Aim number 1 is to determine the oligometric structure of the functional CFTR channel. Specific Aim number 2 is to identify transmembrane (TM) helices that line the pore, by localization of binding sites for open-channel blockers. Specific Aim number 3 is to identify groups of amino acids that serve as determinants of anion selectivity. The proposed approach relies upon the use of molecular biological techniques (site-directed mutagenesis) combined with expression in Xenopus oocytes and quantitative biophysical assays. The working hypothesis is that the pore is lined by TM domains 5, 6, 11, and 12. To achieve these goals, whole-cell and single-channel currents will be measured to determine the kinetics of two structurally-distinct classes of pore-blocking molecules, and to determine whether their binding domains contribute to the permeation pathway. Structural elements that contribute to the architecture of the pore will be defined by comparing the ability of wildtype and mutant channels to interact with open-channel blockers. Previous studies from this laboratory have shown that blocker kinetics are highly sensitive to the structure of the pore. A region within TM6 has been identified that is critical for discrimination between different anions. This region also appears to lie close to the binding sites for pore-blocking molecules. To accurately describe the structure of this region of the channel, it is necessary to consider to contributions made from portions of the channel other than TM6. These studies will be guided by a three- dimensional model of the pore, proposed in the application, which takes into account the experimental data for TM domains 5, 6, 11, and 12. This approach hypothesizes that multiple helical domains contribute both to the binding sites for drugs and to the determinants of selectivity in the channel. A specific subset of residues that may determine the biophysical features of permeation is proposed. Testing the importance of these residues will allow the construction of a detailed map of the conduction pathway. An improved understanding of the function of this channel will aid in the design of novel therapies for Cystic Fibrosis, secretory diarrhea, and polycystic kidney disease.